Confocal microscopes are well known devices. The basic elements of a confocal microscope are illustrated schematically in FIG. 1. In FIG. 1, a light source 1, such as a high-intensity lamp, laser, or light-emitting diode, generates a light beam that passed through a pinhole 2, through dichroic mirror 3, and through objective lens 12, thus forming a spot of focused light on the surface of a sample 10. The objective lens 12 is an aberration-corrected lens.
Light reflected from the surface of the sample 10 (or light generated by a fluorescent or chromophoric dye present in the sample) passes back through the objective lens 12, and is reflected by the dichroic mirror 3 and focused. A pinhole 5 is disposed at the focal point. The focused light coming from the sample, having passed through the pinhole 5, is detected by a photodetector 26.
In a conventional scanning confocal microscope, the sample 10 is rasterized to generate a two-dimensional image of the surface of the sample. To scan the light path across the sample systematically (to thereby generate the rasterized image), a conventional confocal microscope uses a sub-assembly of galvanometers or acousto-optical scanners to manipulate the light path in the X and Y directions. Generally, when using the conventional arrangement shown in FIG. 1, and using galvanometers to scan the light path, the image acquisition rate is about 1 image/sec. If a combination of acousto-optical and galvanometers are used to scan the image, the image acquisition rate is about 30 images/sec. These image acquisition rates limit the usefulness of confocal microscopy because generating a complete image of the sample is too slow.
In an effort to simplify the instrumentation and to speed the image acquisition rate, confocal microscopes wherein the pinhole 5 of FIG. 1. is replaced by a spinning disk having a plurality of apertures passing therethrough are known. In these “rotating disk” confocal microscopes, the single pinhole 5, is replaced by a rotating disk having a pattern of annular apertures therein, the pattern arranged in the shape of an Archimedean spiral. The apertures that are diametrically opposite one another on the disk are on identical radii, and the pattern as a whole has a central symmetry. This type of disk is generally known as a Nipkow disk. Nipkow disks have conventionally been fabricated from a copper foil sheet stretched over a retaining ring and having holes etched into the copper sheet. See, for example, U.S. Pat. No. 4,802,748, issued Feb. 7, 1989, to McCarthy et al.; see also U.S. Pat. No. 3,517,980, issued Jun. 30, 1970, to Petran et al.
For other examples of confocal microscopes utilizing a Nipkow disk, see, for example, U.S. Pat. No. 6,191,885 to Kitagawa, issued Feb. 20, 2001, and U.S. Patent No. 6,204,962 to Kawamura, issued Mar. 20, 2001. These two patents describe a multi-beam optical arrangement wherein a rotating disk scanner is used to scan the laser beam across the sample being viewed. The most significant feature of this type of confocal microscope is that it enables direct observation and direct photography of the sample. In short, this type of multi-beam confocal microscope can be used in the same fashion as a conventional light microscope.
Spinning disk confocal microscopes, however, have several design limitations. Absent complicated realignment of the system, spinning disk confocal microscope designs are restricted to using a single confocal aperture size. Several designs are vulnerable to back-reflection of the excitation light; that is, these designs are prone to having excitation light “leak” into the detection pathway. This is especially so when such a spinning disk device is used in reflected light mode. All disk scanners are limited to using a two-dimensional detector array, such as a CCD camera, to capture the image information. Given these design limitations of the prior art disk scanning confocal instruments, there exists several unmet needs for an instrument that combines the speed and simplicity of the disk scanning devices with the flexibility of the laser scanning systems.